The present invention concerns a method for obtaining mammalian cells, genetically modified (stably transfected) with a fluorescent marker, preferably green fluorescent protein (GFP), as well as mammalian cells with especially high fluorescence.
The use of fluorescent proteins as reporter molecules for gene expression is already known. One such reporter molecules is the green fluorescent protein (GFP). Said marker protein, originally isolated from the jellyfish Aequorea victoria has become an important tool to study gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter molecules. Thus, to improve the detection of GFP in transfected cells GFP derivatives have been developed, such as chromophore mutations and codon optimization for mammalian cells that make the protein e.g. several times brighter than wild-type GFP (see Zhang G. et. al., An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells, Biochem. Biophys. Res. Commun. 227 (3), 707-711 (1996), WO 97/126333 to the University of Florida Research Foundation, Inc. and WO 97/42320 to The Government of the United States of America).
Despite the proven utility of GFP as a reporter molecule for transient gene expression, there still exist problems with the generation of stably transfected cells giving bright signals (see Hanazono Y. et al., Green Fluorescent Protein Retroviral Vectors: Low Titer and High Recombination Frequency Suggest a Selection Disadvantage, Human Gene Therapy 8, 1313-1319 (1997)). The retroviral based method described by Hanazono et al. uses G 418 as a selective drug to isolate stable GFP expressing retroviral producer cell clones, however, the described method was not successful. Gubin A. N. et al. report the successful stable transfection of CHO cells with plasmid DNA encoding both GFP and neomycin phosphor transferase (neo) cassettes (see Gubin A. N. et al., Long term, stable expression of green fluorescent protein in mammalian cells, Biochem. Biophys. Res. Commun., 236(2), 347-350 (1997)). One cloned cell line was reported to maintain xe2x80x9chigh-levelxe2x80x9d green fluorescence for 18 weeks in G 418 and another 12 weeks in non-selective medium. No growth disadvantage in mammalian cells by GFP expression was observed. Stable GFP expression was also obtained in murine cells (see Lybarger L. et al., Rapid Generation and Flow Cytometric Analysis of Stable GFP-Expressing Genes, Cytometry 25, 211-220 (1996)).
The disadvantage of these stably GFP expressing cell lines of the state of the art is their low GFP expression, making them unsuitable for many applications.
A general goal of the present invention is to provide cells, cell lines and/or cell strains, stably expressing fluorescent proteins, that are e.g. producible by the method also being an object of the present invention.
Further objects of the present invention are methods for process development and process optimization wherein the cells/cell lines/cell strains of the present invention are used.
In the following text the term xe2x80x9ccellsxe2x80x9d will be used for cells as well as cell lines and cell strains.
The cells of the present invention are characterized by a fluorescence signal, in particular a fluorescence signal that is at least 100 times the auto-fluorescence of the respective host cell, although preferably at least 200 times higher than said auto-fluorescence and much preferably at least 1000 times said auto-fluorescence. Preferred cells of the present invention are cells, preferably CHO or HEK cells, genetically modified with humanized GFP, in particular those with a fluorescence about 1000 times higher than the auto-fluorescence of the cell.
Suitable proteins for the present invention, herein referred to as xe2x80x9cfluorescent proteinsxe2x80x9d, are all optically measurable proteins that can be measured without specific activation, whereby GFP and its derivatives are preferred proteins.
The process for obtaining cells according to the present invention is characterized by a transfection step that is performed in a way to ensure transient transfection efficiencies of at least 25% positive cells, although preferably about 30% or more, and much preferably 50% or more positive cells after 1 day. The term transient transfection, as it is used in connection with the present invention means an initial state of transfection that is a first step to stable transfection, whereby only a small percentage of transiently transfected cells reach the stable state.
The cells of the present invention arexe2x80x94due to their specific featuresxe2x80x94a very useful tool in process development and optimization making such development and optimization much faster. The fluorescent signal of e.g. GFP can quickly and easily be measured with a standard fluorometer. The signal can be correlated with biomass and/or cell number, extending the application of fluorescent-protein-expressing cells to assays where cell growth is of importance. Information thus obtained allows rapid screening and optimization.